We plan to investigate membrane phospholipid asymmetry in normal and pathologic human erythrocytes. The objectives of our proposal are four-fold: 1. To determine if membrane phospholipid asymmetry in congenital and acquired disorders of the erythrocyte membrane is similar to membrane phospholipid asymmetry in normal erythrocytes. Membrane phospholipid asymmetry will be determined by treatment of intact red cells with both chemical and enzymatic probes which selectively react with phospholipids on the outer lipid leaflet. The effects of metabolic depletion, oxidant injury, and calcium accumulation on the membrane phospholipid asymmetry of normal and abnormal erythrocytes will be compared. 2. To investigate the effects of spectrin and hemoglobin on membrane phospholipid asymmetry. Studies with spectrin-deficient and spectrin-reconstituted mouse erythrocytes will help to define the role of spectrin. Studies on the effects that hemoglobin-phospholipid interactions have on membrane phospholipid asymmetry will be performed using inside out red cell vesicles and lipid-monolayer techniques which permit determination of the interaction between individual phospholipids and hemoglobin variants as well as the effect of spectrin on this interaction. 3. To determine if membrane phospholipid asymmetry has pathophysiologic significance, we will identify erythrocytes with normal and abnormal membrane phospholipid asymmetry and will measure the procoagulant activity of these cells, their ability to fuse in the presence of a fusogen and their adhesion to cultured endothelial cells. 4. To investigate the relationship between membrane phospholipid asymmetry, membrane protein organization, and cellular properties such as shape, cation content, and deformability. We will deliberately perturb membrane phospholipids by treating intact erythrocytes with different phospholipases. After this treatment we will investigate membrane protein organization using cross-linking agents and will measure cellular properties by standard techniques.